This invention is in the field of plant molecular biology. More specifically, this invention pertains to nucleic acid fragments encoding a novel tryptophan synthase beta subunit in plants and seeds.
Many vertebrates, including man, lack the ability to manufacture a number of amino acids and therefore require these amino acids preformed in their diet. These are called essential amino acids. Plants are able to synthesize all twenty amino acids and serve as the ultimate source of the essential amino acids for humans and animals. Thus, the ability to manipulate the production and accumulation of the essential amino acids in plants is of considerable importance and value. Furthermore, the inability of animals to synthesize these amino acids provides a useful distinction between animal and plant cellular metabolism. This can be exploited for the discovery of herbicidal chemical compounds that target enzymes in the plant biosynthetic pathways of the essential amino acids and have low toxicity to animals.
In plants the tryptophan pathway leads to the biosynthesis of many secondary metabolites including the hormone indole-3-acetic acid, antimicrobial phytoalexins, alkaloids and glucosinolates. The two final reactions in tryptophan biosynthesis are catalyzed by tryptophan synthase. The 29 kDa alpha subunit is a bifunctional enzyme which cleaves indole-3-glycerol phosphate to produce indole and glyceraldehyde-3-phosphate. The beta subunit joins indole with serine to form tryptophan. Either subunit alone is enzymatically active, but the rate of the reaction and affinity for the substrates increases when the subunits are forming a tetramer composed of two alpha subunits and two beta subunits (Radwanski (1995) Mol. Gen. Genet. 248:657-667).
Few of the genes encoding enzymes from the tryptophan pathway in corn, soybeans, rice and wheat have been isolated and sequenced. Corn genes encoding tryptophan synthase beta subunits have been identified (Wright et al. (1992) Plant Cell 4:711-719). The instant invention describes novel corn, rice, soybean, and wheat tryptophan synthase beta subunit homologs.
The present invention concerns an isolated polynucleotide comprising a nucleotide sequence selected from the group consisting of: (a) a first nucleotide sequence comprising a polynucleotide of at least 700 nucleotides from SEQ ID NO:1; (b) a second nucleotide sequence comprising a polynucleotide sequence of at least 420 nucleotides from the group consisting of SEQ ID NOs:3, 5, 10, and 12; (c) a third nucleotide sequence encoding a polypeptide of at least 100 amino acids having at least 80% identity based on the Clustal method of alignment when compared to a polypeptide selected from the group consisting of SEQ ID NOs:2, 4, 6, 11, and 13; and (d) a fourth nucleotide sequence comprising a complement of the first or second nucleotide sequences.
In a second embodiment, this invention concerns an isolated polynucleotide comprising a nucleotide sequence of at least 60 (preferably at least 40, most preferably at least 30) contiguous nucleotides derived from a nucleotide sequence selected from the group consisting of SEQ ID NOs:1, 3, 5, 10, and 12 and the complement of such nucleotide sequences.
In a third embodiment, this invention relates to a chimeric gene comprising an isolated polynucleotide of the present invention operably linked to at least one suitable regulatory sequence.
In a fourth embodiment, the present invention concerns a host cell comprising a chimeric gene of the present invention or an isolated polynucleotide of the present invention. The host cell may be eukaryotic, such as a yeast or a plant cell, or prokaryotic, such as a bacterial cell. The present invention also relates to a virus, preferably a baculovirus, comprising an isolated polynucleotide of the present invention or a chimeric gene of the present invention.
In a fifth embodiment, the invention also relates to a process for producing a host cell comprising a chimeric gene of the present invention or an isolated polynucleotide of the present invention, the process comprising either transforming or transfecting a compatible host cell with a chimeric gene or isolated polynucleotide of the present invention.
In a sixth embodiment, the invention concerns a novel plant tryptophan synthase beta subunit polypeptide of at least 100 amino acids comprising at least 80% identity based on the Clustal method of alignment when compared to a polypeptide selected from the group consisting of SEQ ID NOs:2, 4, 6, 11, and 13.
In an seventh embodiment, the invention relates to a method of selecting an isolated polynucleotide that affects the level of expression of a novel plant tryptophan synthase beta subunit polypeptide or enzyme activity in a host cell, preferably a plant cell, the method comprising the steps of: (a) constructing an isolated polynucleotide of the present invention or a chimeric gene of the present invention; (b) introducing the isolated polynucleotide or the chimeric gene into a host cell; (c) measuring the level of the novel plant tryptophan synthase beta subunit polypeptide or enzyme activity in the host cell containing the isolated polynucleotide; and (d) comparing the level of the novel plant tryptophan synthase beta subunit polypeptide or enzyme activity in the host cell containing the isolated polynucleotide with the level of the novel plant tryptophan synthase beta subunit polypeptide or enzyme activity in the host cell that does not contain the isolated polynucleotide.
In a eighth embodiment, the invention concerns a method of obtaining a nucleic acid fragment encoding a substantial portion of a novel plant tryptophan synthase beta subunit polypeptide, preferably a plant novel plant tryptophan synthase beta subunit polypeptide, comprising the steps of: synthesizing an oligonucleotide primer comprising a nucleotide sequence of at least 60 (preferably at least 40, most preferably at least 30) contiguous nucleotides derived from a nucleotide sequence selected from the group consisting of SEQ ID NOs:1, 3, 5, 10, and 12 and the complement of such nucleotide sequences; and amplifying a nucleic acid fragment (preferably a cDNA inserted in a cloning vector) using the oligonucleotide primer. The amplified nucleic acid fragment preferably will encode a substantial portion of a novel plant tryptophan synthase beta subunit amino acid sequence.
In a ninth embodiment, this invention relates to a method of obtaining a nucleic acid fragment encoding all or a substantial portion of the amino acid sequence encoding a novel plant tryptophan synthase beta subunit polypeptide comprising the steps of: probing a cDNA or genomic library with an isolated polynucleotide of the present invention; identifying a DNA clone that hybridizes with an isolated polynucleotide of the present invention; isolating the identified DNA clone; and sequencing the cDNA or genomic fragment that comprises the isolated DNA clone.
In a tenth embodiment, this invention concerns a composition, such as a hybridization mixture, comprising an isolated polynucleotide or an isolated polypeptide of the present invention.
In an eleventh embodiment, this invention concerns a method for positive selection of a transformed cell comprising: (a) transforming a host cell with the chimeric gene of the present invention or a construct of the present invention; and (b) growing the transformed host cell, preferably a plant cell, such as a monocot or a dicot, under conditions which allow expression of the novel plant tryptophan synthase beta subunit polynucleotide in an amount sufficient to complement a null mutant to provide a positive selection means.
In a twelfth embodiment, this invention relates to a method of altering the level of expression of a novel plant tryptophan synthase beta subunit in a host cell comprising: (a) transforming a host cell with a chimeric gene of the present invention; and (b) growing the transformed host cell under conditions that are suitable for expression of the chimeric gene wherein expression of the chimeric gene results in production of altered levels of the novel plant tryptophan synthase beta subunit in the transformed host cell.
A further embodiment of the instant invention is a method for evaluating at least one compound for its ability to inhibit the activity of a novel plant tryptophan synthase beta subunit, the method comprising the steps of: (a) transforming a host cell with a chimeric gene comprising a nucleic acid fragment encoding a novel plant tryptophan synthase beta subunit polypeptide, operably linked to at least one suitable regulatory sequence; (b) growing the transformed host cell under conditions that are suitable for expression of the chimeric gene wherein expression of the chimeric gene results in production of the tryptophan synthase beta subunit in the transformed host cell; (c) optionally purifying the novel plant tryptophan synthase beta subunit polypeptide expressed by the transformed host cell; (d) treating the novel plant tryptophan synthase beta subunit polypeptide with a compound to be tested; and (e) comparing the activity of the novel plant tryptophan synthase beta subunit polypeptide that has been treated with a test compound to the activity of an untreated novel plant tryptophan synthase beta subunit polypeptide, thereby selecting compounds with potential. for inhibitory activity.